Scientific Objectives and Approaches: A substantial portion of the plant proteome is localized in the cell wall/apoplast. This is not surprising, given the central role of the wall in many aspects of plant biology, including cell growth and differentiation, defence, metabolism and transport of nutrients and responses to environmental stresses. However, the complement of proteins that are secreted to the wall, or "secretome", is far less characterized than those of other plant subcellular compartments. This proposal outlines an integrated approach to catalog the tomato secretome, coupling a suite of newly developed functional screens and sequencing of highly purified wall protein extracts, with bioinfomatic tools and computational prediction. This infrastructure will be used to evaluate the qualitative and quantitative dynamics of the wall proteome during two biological processes that are intimately associated with wall biology: fruit ripening and plant defence responses. The specific objectives of this project are:

Apply complementary experimental and computational strategies to catalog the plantsecretome in a range of tomato tissues

Develop a bioinformatics tool to allow a more accurate in silico prediction of the secretome

Characterize the quantitative and qualitative dynamics of the wall proteome and wallphosphoproteome, using Difference Gel Electrophoresis (DIGE) and shotgun sequencing of isotopically labeled proteins (iTRAQ) in ripening fruit and in leaves following elicitation of defence response

Contrast the changes in the secretome of ripening wild type tomato fruit with corresponding changes in the wall-associated transcriptome of wild type and ripening impaired mutant fruit

Create a publicly accessible tomato secretome database (SecreTom), to house the data generated through the proposed research, to provide access to all associated computational tools and to act as a current and future hub for plant wall proteome research

An important element of this project is the development of a strong education and training component. In addition to the expected training of graduate and undergraduate students, this will take the form of two new programs: (a) a Plant Genome Research internship program that has been created as a Cornell-wide initiative to bring together a critical mass of undergraduate students, high school students and high school teachers on the Cornell campus each summer. These individuals will participate in lab-based research and mentoring within the larger genomics-related community of plant scientists; (b) an annual summer Proteomics Workshop, comprising a seminar series, poster-presentations and lab-based practical training. In both cases, the participation of under-represented groups at the undergraduate and graduate levels will be ensured through the support of a network of existing Cornell programs. The project will generate a valuable, publicly available resource in the form of a webbased plant secretome database thus ensuring rapid public dissemination of the data. Importantly, this proposal will take full advantage of, and enhance, previous investments by the NSF in tomato genomics and provide a template for similar studies in other plant species, many of which are agriculturally and commercially important.